3.1 Rational design of FGF2 variants
Selection of an appropriate modification site is important for the
retention of biological activity of PEGylated proteins. The surface of
FGF2 contains two native cysteines, Cys69 and Cys87, which are surface
exposed and spatially distal from both the receptor and heparin binding
regions. We separately mutated each cysteine to alanine, thus generating
two FGF2 containing only a single surface-exposed cysteine (compounds1 and 2 ). And two surface-exposed residues, Lys129 and
Phe17, near the heparin- and receptor-binding regions, respectively,
were also changed to cysteines (compounds 3 and 4 )
(Figure 1A ). The resulting FGF2 variants, each containing a
single cysteine for PEGylation, were engineered and tested:
FGF2C69A (compound 1 ),
FGF2C87A (compound 2 ),
FGF2C69A/C87A/F17C (compound 3 ), and
FGF2C69A/C87A/K129C (compound 4 )
(Figure 1 B and E ).
The activation of extracellular signal-regulated kinases 1 and 2
(ERK1/2) is a key point of convergence for many signaling pathways, and
the biological response of extracellular stimuli including FGF2 requires
ERK1/2 activation (Johnson et al. , 2002). Hence, signaling of the
ERK1⁄2 pathway was evaluated by immunoblotting to assay the activity of
FGF2 and its mutants. The cellular phosphorylated ERK1⁄2 (p-ERK1/2)
levels were increased with native FGF2 compared with the control group,
and the same increase occurred with compounds 1 and 2(Figure 1 C-D ). A slight
reduction in the cellular level of p-ERK1⁄2 was observed for compounds3 and 4 (Figure 1 E-F ). These results
indicated that both the receptor- and heparin-binding regions are indeed
important for FGF2 activity and that single site mutation distal from
the binding domains has little effect on protein activity.